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D A Bushinsky

Publications and source records attributed to D A Bushinsky.

At least 91 records · Page 5Linked to original sources

Effects of parathyroid hormone on net proton flux from neonatal mouse calvariae.

Bone mineral buffers protons during acute metabolic acidosis; whether parathyroid hormone (PTH) augments proton buffering is controversial. To determine whether PTH augments proton buffering by bone, we cultured neonatal mouse calvariae with or without PTH (10(-8) M) for 3 h in medium that was physiologically acid (pH approximately 7.20), neutral (pH approximately 7.40), or alkaline (pH approximately 7.60). Over the entire pH range studied there was less influx of protons into calvariae treated with PTH than into control calvariae, indicating that PTH does not augment but instead inhibits proton buffering by bone. To determine whether chronic exposure to PTH is necessary to augment proton buffering, calvariae were incubated with PTH for 24 h before a 3-h culture. Calcium efflux from calvariae exposed to PTH (10(-8) M) for 24 h exceeded that of controls. When these same calvariae were recultured for 3 h in fresh medium, PTH-treated and control calvariae behaved similarly, with net efflux of protons into acid, neutral, and alkaline media. Regardless of whether PTH is added at the time of exposure to acid medium or 24 h before calvariae cultured with PTH do not buffer protons to a greater extent than controls.

Acidosis↗

Mechanism of proton-induced bone calcium release: calcium carbonate-dissolution.

Protons are buffered and calcium is released by bone during metabolic acidosis. Incubation of neonatal mouse calvariae in acid medium causes net calcium efflux from bone and net proton influx into bone, just as metabolic acidosis does in vivo. To determine whether the calcium carbonate phase of bone mineral is solubilized with increasing proton concentrations, we cultured calvariae for 3 h in medium in which the saturation was varied by changing pH or calcium and phosphate concentrations. We determined the driving force for crystallization by calculating the Gibbs free energy of formation (DG). With alteration of the medium pH, calcium carbonate entry or loss from bone varied linearly with the initial DG for medium calcium carbonate (r = -0.745, n = 41, P less than 0.001) as it did with alteration of the medium calcium and phosphate (r = -0.665, n = 118, P less than 0.001). There was dissolution of calcium carbonate into medium that was unsaturated with respect to calcium carbonate, net flux ceased at saturation, and calcium carbonate entered bone from supersaturated medium, indicating that the medium is in equilibrium with the calcium carbonate phase of bone mineral. Neither the mineral phase brushite nor apatite was in equilibrium with the medium. These observations indicate that in vitro, acute proton-induced calcium efflux is due to dissolution of bone calcium carbonate.

Acidosis↗

Effects of medium pH on duodenal and ileal calcium active transport in the rat.

To study the effect of pH on Ca active transport in vitro pH was varied from 7.1 to 7.7, and bidirectional transmural Ca fluxes were measured under short-circuited conditions across duodenum or ileum from rats fed either normal chow (NCD, 1.2% Ca) or low Ca (LCD, 0.002% Ca). Duodenum and ileum from rats fed LCD actively absorbed calcium at medium pH 7.4. Reduction in mucosal and serosal medium bicarbonate from 25 to 10 mM (pH 7.4 to 7.1) decreased duodenal net Ca absorption (Jnet) from 121 +/- 32 to 39 +/- 9 nmol X cm-2 X h-1 (P less than 0.02) and ileal Jnet from 74 +/- 13 to 22 +/- 6 (P less than 0.01). The decline in duodenal and ileal Ca Jnet was due to a decrease in the mucosal-to-serosal flux (Jm----s). Raising medium pH from 7.4 to 7.7 by increasing bicarbonate from 25 to 50 mM did not alter Ca Jm----s, serosal-to-mucosal flux (Js----m), or Jnet X Ca fluxes across ileum from rats fed NCD demonstrated net secretion. LCD ileal Jm----s was unaltered when pH was reduced from 7.4 to 7.1 by increasing the PCO2 content of the buffer from 30 to 81 mmHg while maintaining bicarbonate at 24 mM. The results indicate that in vitro conditions that simulate metabolic acidosis (low bicarbonate and pH, normal PCO2) inhibit 1,25D-mediate calcium Jm----s, whereas conditions that simulate respiratory acidosis have no effect on Ca fluxes. The present studies suggest that decreases in calcium Jm----s is by a primary alteration in transport of other ions rather than direct effect on Ca transport.

Animals↗

Ion microprobe determination of bone surface elements: effects of reduced medium pH.

When neonatal mouse calvariae are cultured in a medium having a low pH they release calcium (Ca) while buffering protons (H). However, for 1 neq Ca released, 16-21 neq H enter the calvariae, arguing strongly against simple dissolution of bone mineral as the mechanism of H buffering. To determine if H for sodium (Na) exchange could explain the lack of stoichiometry between H and Ca, we cultured calvariae for 3 h in control (pH = 7.40 +/- 0.01) or reduced-pH (pH = 7.21 +/- 0.01) medium and then examined the surface Na, K, and Ca using a high-resolution scanning ion microprobe. The calvarial surface was rich in Na and K relative to Ca (Na/Ca, 52 +/- 17; K/Ca, 61 +/- 17; all values are the ratios of counts per second of detected secondary ions, means +/- SE). Compared with the surface the calvarial cross section contained far less Na and K relative to Ca (Na/Ca, 2 +/- 1; K/Ca, 1 +/- 1; both P less than 0.01 vs. surface). Compared with the control surface, culture in a reduced-pH medium reduced the surface Na and K relative to Ca (Na/Ca, 5 +/- 1; K/Ca, 7 +/- 1; both P less than 0.025 vs. surface) to values still greater than the cross section (P less than 0.05 for both). Neonatal mouse calvariae have a surface that is rich in Na and K relative to Ca. Reduced medium pH depletes surface Na and K of cultured calvariae with respect to Ca.

Acidosis↗

Mechanism of chronic hypercalciuria with furosemide: increased calcium absorption.

Furosemide produces chronic hypercalciuria. The source of the additional urinary calcium is not known but must be either bone mineral or calcium absorbed by the intestine. Without bone calcium dissolution or increased absorption the filtered load of calcium would fall and urinary calcium excretion would return to pretreatment levels. To determine whether furosemide alters intestinal calcium absorption, we fed furosemide (75 mg . kg body-1 wt . day-1) to 11 rats eating 15 g/day of a 0.60% calcium diet. Compared with 11 control rats, furosemide increased urine calcium (15.6 +/- 0.8 mg/5 days vs. 4.1 +/- 0.3, P less than 0.001). Fecal calcium excretion fell (194 +/- 7 mg/5 days vs. 223 +/- 12, P less than 0.05), indicating an increase in intestinal calcium absorption sufficient to sustain the hypercalciuria. The increase in absorption occurred without an increase in the level of serum 1,25-dihydroxycholecalciferol (180 +/- 20 pg/ml vs. 220 +/- 16, furosemide vs. control, respectively, P = NS). To determine whether the intestinal effect of furosemide persists after the initial sodium diuresis abates, we analyzed only the last 3 days of balance. Again, rats fed furosemide had increased urine excretion and intestinal absorption of calcium, so that net calcium balance was not different from that of controls. Twelve additional rats were fed a 0.02% calcium diet to which 35 mg . kg body wt-1 . day-1 of furosemide was added. Compared with eleven controls, urine calcium increased and fecal calcium excretion again fell, but balance was not different. Chronic administration of furosemide increases intestinal calcium absorption enough to permit urine calcium excretion to remain elevated without the necessity for bone dissolution.

Animals↗

Ca and P regulation of 1,25(OH)2D3 synthesis by vitamin D-replete rat tubules during acidosis.

Elevated blood ionized Ca concentration appears to be necessary for suppression by chronic metabolic acidosis (CMA) of serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels in vitamin D-replete rats eating a low-Ca diet (LCD). The present study asks whether in vitro 1,25(OH)2D3 production by proximal tubule cells from such rats is suppressed and whether suppression can be reversed in vitro by an altered ionic milieu. Young rats were fed LCD and given 1.5% NH4Cl in their drinking water for 10 days to produce CMA. Compared with controls, CMA rats had low serum 1,25(OH)2D3 levels. Tubules prepared from CMA rats produced 1,25(OH)2D3 at a low rate compared with control; variation of medium Ca and pH from 7.2 to 7.4 did not increase 1,25(OH)2D3 production. Reducing medium phosphorus concentration ([P]) increased 1,25(OH)2D3 production by tubules from control but not CMA rats. Increasing medium [P] increased 1,25(OH)2D3 production by tubules from CMA but not control rats. CMA appears to alter proximal tubule cell response to medium [P] so that 1,25(OH)2D3 production rises when medium [P] is increased but not when it is reduced. Medium pH and Ca concentration do not appear to be important regulators of renal 1,25(OH)2D3 production in short-term incubations.

Acidosis↗

Evidence that serum calcium oxalate supersaturation is a consequence of oxalate retention in patients with chronic renal failure.

Serum oxalate rises in uremia because of decreased renal clearance, and crystals of calcium oxalate occur in the tissues of uremic patients. Crystal formation suggests that either uremic serum is supersaturated with calcium oxalate, or local oxalate production or accumulation causes regional supersaturation. To test the first alternative, we ultrafiltered uremic serum and measured supersaturation with two different methods previously used to study supersaturation in urine. First, the relative saturation ratio (RSR), the ratio of the dissolved calcium oxalate complex to the thermodynamic calcium oxalate solubility product, was estimated for 11 uremic (before and after dialysis) and 4 normal serum samples using a computer program. Mean ultrafiltrate oxalate predialysis was 89 +/- 8 microM/liter (+/- SEM), 31 +/- 4 postdialysis, and 10 +/- 3 in normals. Mean RSR was 1.7 +/- 0.1 (predialysis), 0.7 +/- 0.1 (postdialysis), and 0.2 +/- 0.1 (normal), where values greater than 1 denote supersaturation, less than 1, undersaturation. Second, the concentration product ratio (CPR), the ratio of the measured calcium oxalate concentration product before to that after incubation of the sample with calcium oxalate monohydrate crystal, was measured in seven uremic and seven normal serum ultrafiltrates. Mean oxalate was 91 +/- 11 (uremic) and 8 +/- 3 (normal). Mean CPR was 1.4 +/- 0.2 (uremic) and 0.2 +/- 0.1 (normal). Predialysis, 17 of 18 uremic ultrafiltrates were supersaturated with respect to calcium oxalate. The degree of supersaturation was correlated with ultrafiltrate oxalate (RSR, r = 0.99, r = 29, P less than 0.001; CPR, r = 0.75, n = 11, P less than 0.001). A value of ultrafiltrate oxalate of 50 microM/liter separated undersaturated from supersaturated samples and occurred at a creatinine of approximately 9.0 mg/dl.

Adult↗

Cellular contribution to pH-mediated calcium flux in neonatal mouse calvariae.

Net calcium flux from cultured neonatal mouse calvariae into the culture medium is pH dependent, and acidified culture medium causes egress of calcium from bone. To determine whether calcium flux is mediated by pH effects on bone cell function, we cultured calvariae for 24 h with sodium azide, acetazolamide, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or after three successive freeze-thaw cycles, treatments that would be expected to alter bone cell function. We recultured bones for 3 h with the respective treatment and measured calcium flux. Sodium azide and freeze-thaw cycles produced a net influx of calcium (JCa = -22 +/- 7 and -23 +/- 6 nmol X bone-1 X 3 h-1, respectively) compared with net efflux of control bones (JCa = 35 +/- 6) at a similar initial medium pH. Acetazolamide reduced net flux to 0 (JCa = 7 +/- 6). PTH and 1,25(OH)2D3 increased net calcium efflux from bone (JCa = 78 +/- 7 and 74 +/- 10, respectively). Despite changing net flux, the slope dependence of net flux on medium pH was the same in the control group and all five treated groups of bones. The similarity of slopes indicates that the pH dependence of net flux is not a result of pH acting on bone cells but probably an effect of altered mineral equilibria. The difference in net flux at similar pH indicates that calcium efflux is partially inhibited by acetazolamide and stimulated by both PTH and 1,25(OH)2D3.

Acetazolamide↗

Response of serum 1,25(OH)2D3 to variation of ionized calcium during chronic acidosis.

Chronic ammonium chloride (NH4Cl) administration causes metabolic acidosis and prevents the normal rise of serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] during a low-calcium diet (LCD, 0.002% calcium). The suppression of serum 1,25(OH)2D3 was not due to reduced parathyroid hormone concentration, elevated serum phosphorus, or total calcium concentration. Acidosis increased blood ionized Ca [Ca2+] and proton [H+] concentrations. Serum 1,25(OH)2D3 levels were inversely correlated with both [Ca2+] and [H+]. To determine the independent effects of [Ca2+] on serum 1,25(OH)2D3 we varied [Ca2+] at a constant [H+] by infusing either EGTA or saline for 24 h after 11 days of LCD and NH4Cl. EGTA, preequilibrated with three concentrations of Ca, lowered [Ca2+] and raised 1,25(OH)2D3 but did not alter [H+] or serum phosphorus concentration. The log of serum 1,25(OH)2D3 varied linearly and inversely with arterial blood [Ca2+] during saline (r = -0.884, n = 8, P less than 0.001) and EGTA infusions (r = -0.798, n = 22, P less than 0.001). At all levels of [Ca2+], rats infused with EGTA had a higher serum 1,25(OH)2D3 than those infused with saline. Log serum 1,25(OH)2D3 was correlated neither with [H+] nor pH. Elevated [Ca2+] and not [H+] appears to suppress the serum 1,25(OH)2D3 response to LCD during NH4Cl acidosis in the rat.

Acidosis↗

Hyperkalemia during acute ammonium chloride acidosis in man.

The relationship between acid base parameters and serum potassium concentration was studied in controls and in patients during acute ammonium chloride-induced metabolic acidosis. Serum potassium was best correlated with serum bicarbonate during control (r = -0.323; p less than 0.01) and acidosis (r = -0.437; p less than 0.001). The slopes and intercepts were similar in both instances and the combined correlation was highly significant (r = -0.493; p less than 0.001). Examination of the 95% joint confidence region revealed that during acidosis serum potassium was rarely above 5.0 mEq/l when serum bicarbonate was greater than 16 mEq/l. It is probably not sound clinical practice to ascribe hyperkalemia to acute mild metabolic acidosis.

Acidosis↗

Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat.

This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.

Animals↗

Effects of dietary calcium restriction on 1,25-dihydroxyvitamin D3 net synthesis by rat proximal tubules.

We measured in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by kidney proximal tubules prepared by Percoll density centrifugation from male and female rats. 1,25(OH)2D3 in tubule extracts was determined by a sensitive and specific radioreceptor assay. Ingestion of diets adequate in vitamin D3 and containing either normal calcium (1.2% Ca, NC), reduced calcium (0.6% Ca, RCD) or low calcium (0.002% Ca, LCD) increased 1,25(OH)2D3 net synthesis (for male rats, NC vs. RCD vs. LCD 1.8 +/- 0.1 SEM vs. 9 +/- 2 vs. 17 +/- 2 pmol/mg protein/20 min; P less than 0.05 for all comparisons). At either level of reduced calcium intake, tubules from male rats produced more 1,25(OH)2D3 than tubules from females. Serum 1,25(OH)2D3 and tubule cyclic adenosine monophosphate (cAMP) content rose in parallel with progressive dietary calcium restriction, and males had higher circulating 1,25(OH)2D3 and tubule cAMP content than females at each level of reduced calcium intake. L-Epinephrine (10(-4) mol/L), in vitro, increased tubule accumulation of 1,25(OH)2D3 and cAMP. Yohimbine, and alpha 2-receptor antagonist, blocked this response, whereas prazosin was without effect. Increased 1,25(OH)2D3 net synthesis by tubules from male vs. female rats partly explains the higher serum levels and enhanced mineral conservation demonstrated previously in male rats. Preparation of proximal tubules from vitamin D-replete rats permits studies in vitro of 1,25(OH)2D3 production and regulation under more physiologic conditions in which parathyroid hormone, inorganic phosphorus, and calcium may be varied independently.

Animals↗

Elevated 1,25(OH)2D3, intestinal absorption, and renal mineral conservation in male rats.

We studied the physiological importance of elevated serum 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] in male rats. Male and female rats of equal weight consumed either a high calcium diet (1.20% calcium; HCD), a low calcium diet (0.02% calcium; LCD), or a normal calcium diet (0.60% calcium; NCD). With all three diets, serum 1,25(OH)2D3 was higher in males than females and rose in both sexes with decreasing diet calcium. There was a direct correlation between serum 1,25(OH)2D3 and proximal duodenal 45Ca uptake (r = 0.667; P less than 0.001). With NCD, we performed a balance study and found a direct correlation between serum 1,25(OH)2D3 and both 5-day fractional calcium absorption (r = 0.703; P less than 0.01) and fractional phosphorus absorption (r = 0.679; P less than 0.01). In addition, serum 1,25(OH)2D3 was directly correlated with 5-day calcium retention (r = 0.772; P less than 0.001) and phosphorus retention (r = 0.830; P less than 0.001). Males grew faster than females (F ratio = 1,194; P less than 0.001). Elevated 1,25(OH)2D3 in males appears to be of biological significance and may help satisfy a greater, growth-related, need for calcium.

Absorption↗

Pathophysiology of hypercalciuria.

The mechanisms responsible for hypercalciuria may involve intestinal calcium transport, renal tubule calcium reabsorption, and the regulation of bone mineral content. Both parathyroid hormone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) may alter urine calcium. For these reasons, understanding the pathogenesis of hypercalciuria in patients has proven to be difficult. We present here an analysis of pathways that regulate systemic calcium homeostasis and of the various mechanisms that have been proposed to explain normocalcemic hypercalciuria in humans. Available evidence seems to implicate disordered regulation of 1,25(OH)2D3 as a basis for at least one common form of hypercalciuria.

Benzothiadiazines↗

Mechanism of chronic hypocalciuria with chlorthalidone: reduced calcium absorption.

Chlorthalidone, like other benzothiadiazides, lowers urine calcium excretion chronically. If intestinal calcium absorption did not fall or bone accretion did not increase, serum calcium and the filtered load of calcium would increase and urine calcium would return to pretreatment levels. To determine whether overall intestinal calcium absorption fell, we fed chlorthalidone (5 mg X kg body wt-1 X 24 h-1) to 10 adult male rats eating 15 g/day of a 0.6% calcium diet. Compared with 10 control rats, chlorthalidone reduced urine calcium [2.1 +/- 0.1 (SE) vs. 5.8 +/- 0.5 mg/6 days; P less than 0.001]. Fecal calcium rose (307 +/- 9 vs. 257 +/- 12; P less than 0.005) because percent intestinal calcium absorption fell (41 +/- 2 vs. 52 +/- 2; P less than 0.002). Twenty other rats given the same diet were injected subcutaneously with 1,25(OH)2D3 (50 ng/day). In these rats, chlorthalidone reduced urine calcium (23 +/- 3 vs. 59 +/- 3; P less than 0.001) and percent intestinal calcium absorption (60 +/- 1 vs. 66 +/- 1; P less than 0.01). With or without 1,25(OH)2D3, chronic administration of chlorthalidone reduces intestinal calcium absorption, and this reduction seems to be the mechanism that permits urine calcium excretion to remain low.

Animals↗

Hospital-acquired renal insufficiency: a prospective study.

Twenty-two hundred sixty-two consecutive medical and surgical admissions were evaluated prospectively to determine the contribution of iatrogenic factors to the development of renal insufficiency in hospital. Of 2,216 patients at risk, some degree of renal insufficiency developed in 4.9 percent. Decreased renal perfusion, postoperative renal insufficiency, radiographic contrast media, and aminoglycosides accounted for 79 percent of the episodes. Iatrogenic factors, broadly defined, accounted for 55 percent of all episodes. Poor prognostic indicators included oliguria, urine sediment abnormalities and, most importantly, severity of renal insufficiency; with an increase in serum creatinine of 3 mg/dl or greater, the mortality rate was 64 percent. Age, admission serum creatinine levels, and the number of episodes of renal insufficiency did not significantly affect outcome. We conclude that there is a substantial risk of the development of renal failure in hospital and that the mortality rate due to hospital-acquired renal insufficiency remains high.

Acute Kidney Injury↗

Effects of pH on bone calcium and proton fluxes in vitro.

Bone mineral is thought to decompose during acute and chronic metabolic acidosis and thereby contribute to buffering of the acid load. We cultured neonatal mouse calvariae for 3 h and found calcium efflux from bone when the medium pH was below 7.40, calcium influx into bone when the pH was above 7.40, and no net flux at pH 7.40. The calcium flux varied to the same extent when medium pH was altered by a primary change in the medium bicarbonate concentration or in the partial pressure of carbon dioxide. Calcium and proton fluxes were inversely correlated (r = -0.713, P less than 0.001), and the slope of the linear regression indicated that between 16 and 21 neq of proton entered the calvariae in exchange for each neq of calcium that left. In 24-h cultures, acid medium also caused net calcium efflux from bone, and alkaline medium caused net influx. PTH increased calcium efflux at acid but not at alkaline medium pH. Sodium azide resulted in net influx of calcium into bone at all values of medium pH. Calcium release by cultured calvariae in response to low medium pH is associated with proton buffering; over 3 h the stoichiometry indicates that little buffering is due to the dissolution of calcium-containing crystals. Effects of medium pH on calcium release are amplified by PTH, and calcium efflux can be prevented by the metabolic inhibitor sodium azide.

Animals↗